This application addresses broad Challenge Area (11) Regenerative Medicine, and specific Challenge topic, 11-EB-104: Living Human Tissue Microarrays, aimed at generating "organotypic platforms that are complex yet modular, hardened, standardized, simplified, and validated against traditional animal models." Animal studies are proving to be insufficient for predicting human liver responses primarily due to significant species-specific differences in liver functions. Therefore, a plethora of in vitro human liver models have been developed over the last three decades to supplement testing on animals. Of the several liver models currently available, those utilizing primary hepatocytes strike a good balance between their potential to predict the diverse human responses seen in vivo and their simplicity of use in various culture formats. However, primary hepatocytes are notoriously difficult to maintain in conventional models as their phenotypic functions display a precipitous decline within a few hours after isolation from the native microenvironment of the liver. Indeed, unstable hepatocytes in these models have been shown to be poor predictors of clinical outcomes. We have utilized microfabrication technologies and tissue engineering techniques to develop a human liver model with precise microscale cytoarchitecture and optimal stromal interactions that displays phenotypic stability for several weeks in vitro as compared to a few hours in conventional cultures. Here, we propose to further develop and optimize these microscale human liver cultures and couple them with miniaturization strategies and assay technologies for cost-effective high-throughput screening (HTS) applications. Since drug-induced liver injury (DILI) is a leading cause of acute liver failures and the high attrition rate of pharmaceuticals, we will optimize our miniaturized human livers specifically for the in vitro screening of genotype-specific and clinically- relevant drug disposition and coupled DILI. The technologies we develop here may find broad utility in the development of several classes of therapeutic compounds (drugs, biologics), in evaluating the disposition and injury potential of environmental toxicants, in fundamental investigations of liver physiology and disease, and in personalized medicine for liver disease. In the future, continued combination of microtechnology with tissue engineering may spur the development of other tissue models and their integration into the so-called 'human- on-a-chip'. PUBLIC HEALTH RELEVANCE: The studies proposed in this project are aimed towards developing a miniaturized human liver microarray for high-throughput screening applications, specifically for evaluating drug disposition and drug- induced liver injury, a serious challenge for patients, regulatory agencies and the pharmaceutical/biotech industry. In the future, our miniaturized human liver HTS system may eliminate problematic compounds much earlier in the drug development pipeline towards reducing patient exposure to unsafe drugs. The technologies we develop here may also find utility in assessing the injury potential of environmental toxicants, in basic research, and in personalized medicine for patients with liver disease.